The present invention generally relates to the fields of nucleic acid analogs and hybridization. More specifically, the invention relates to methods and compositions for hybridization of a collection of probes to a target nucleic acid.
Nucleic acids, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), are bearers of information. This information, encoded in the ordered nucleotides that constitute the nucleic acids, enables a living system to construct a protein, a cell, or an organism. One specific sequence of nucleotides may be found in a virus, whereas a different sequence may be found in a bacterium, and yet a different sequence in a human being. The detection and analysis of nucleic acids has become one of the most fundamental aspects of diagnostic medicine and medical research. Because nucleic acid sequences can also differ among individuals, nucleic acid analysis has also become important to forensic medicine.
Most current methods of nucleic acid analysis require the hybridization of one or more oligonucleotides to a target nucleic acid of interest. The hybridization step is often followed by an enzymatic reaction involving the addition of nucleotides to the hybridized oligonucleotide, as in primer extension reactions or in polymerase chain reactions (PCR). In other cases, the hybridization step is followed by washing and detection steps, as in Southern or Northern blot analysis.
The hybridization of an oligonucleotide to a target nucleic acid generally requires that the sequence of the oligonucleotide be approximately complementary to the sequence of the target nucleic acid. Thus, after the sequence of the target nucleic acid of interest is determined, an appropriate, complementary oligonucleotide for use in hybridization to the target nucleic acid must be designed.
In most cases, the complementary oligonucleotide must be custom-synthesized. Most oligonucleotides used for this purpose are at least twelve nucleotides in length to permit efficient hybridization. Because any of four nucleotides could be present at each position in the oligonucleotide, there are 412 or 16,777,216 possible oligonucleotides that are twelve nucleotides in length. The skilled artisan is therefore unlikely to possess, in advance, a newly-desired oligonucleotide. Unfortunately, custom synthesis of oligonucleotides is both expensive and time-consuming: the process may require from 3-6 business days, including ordering, synthesis, and shipping. Inevitably, analysis of the nucleic acids is further delayed.
Thus, because of the importance of nucleic acid analysis to modem medical science, there is a great need for faster, cheaper, and more reliable means to carry out this analysis, such as methods that do not depend on custom-synthesized oligonucleotides. Similarly, there is a need for nucleic acid binding moieties that can be generated rapidly and inexpensively without the impediments of traditional custom synthesis.
It has been discovered that a nucleic acid binding moiety can be generated by combining two or more smaller probes that interact to generate a single binding moiety, also referred to herein as a multipartite binding moiety. For example, if one of the smaller probes includes a portion complementary to a first nucleic acid sequence (xe2x80x9cXxe2x80x9d), and another of the smaller probes includes a portion complementary to a second nucleic acid sequence (xe2x80x9cYxe2x80x9d), then a new, single binding moiety would include a composite nucleic acid recognition sequence complementary to the combination of the first and second regions (X+Y). Accordingly, the invention allows one skilled in the art to create a probe complementary to a target nucleic acid sequence by combining smaller probes, each of which is complementary to a particular subset of the target nucleic acid sequence and is capable of interacting with the other smaller probe(s). This interaction can be achieved, for example, through the formation of a three-way junction.
It has been discovered that a three-way junction can be stabilized by the introduction of a flexible linker between the portion of the smaller probe that interacts with the target nucleic acid and the portion that interacts with the other smaller probe(s). It has also been discovered that the use of peptide nucleic acids or other nucleic acid analogs that interact more strongly with a strand of DNA than would the complementary strand of DNA can improve the affinity of the smaller probes for each other and for the target nucleic acid. Thus, a stable interaction is possible even where each of the smaller probes is complementary only to a small region of the target nucleic acid (e.g. three to eight nucleotides).
One aspect of the invention is a collection of at least two probes for use in hybridizing to a target nucleic acid. In this aspect, a first probe includes a first portion that may be complementary to a first region of a target nucleic acid and capable of hybridizing thereto, joined by a flexible linker to a second portion capable of hybridizing with the second probe. Similarly, the second probe includes a first portion that may be complementary to a second region of the target nucleic acid and capable of hybridizing thereto, and a second portion capable of hybridizing with the first probe. Both the first and second regions of the target nucleic acid typically are from three to eight nucleotides in length, and preferably substantially adjacent, i.e. separated by zero or one nucleotides. Either the first probe or the second probe, or both, is or includes a high-affinity nucleic acid analog. A preferred high-affinity nucleic acid analog is PNA (peptide nucleic acid), where the sugar/phosphate backbone of DNA or RNA has been replaced with a polyamide backbone, e.g. 2-aminoethylglycine.
Because this invention provides a means to generate a larger probe by combining two or more smaller probes, in other embodiments of the invention, the collection of probes includes an array of a plurality of first probes (a library of first probes) and an array of a plurality of second probes (a library of second probes) which may be used to generate a larger probe. In these embodiments, the portion of each of the first probes that may be complementary to the first region of the target nucleic acid has a different sequence, and the portion of each of the second probes that may be complementary to the second region of the target nucleic acid has a different sequence.
The array of first probes often includes at least 50% of the possible combinations of first probes. Thus, if the first region of the target nucleic acid is x nucleotides in length and if the portion of each of the first probes that may be complementary to that region is non-degenerate, the array of first probes includes at least 0.5xc3x974x first probes. Likewise, the array of second probes often includes at least 50% of the possible combinations of second probes.
In other embodiments, the collection of probes is provided in a kit, in combination with a buffer. In one preferred embodiment, the kit also includes an enzyme. In another preferred embodiment, the kit also includes a detection moiety.
Another aspect of the invention is a method of using the collection of probes of the invention. In one embodiment, the invention is a method of detecting the presence of a target nucleic acid sequence. Generally, the target nucleic acid is exposed to the first and second probes to form a complex if the target nucleic acid sequence is present. The presence or absence of the complex may be determined by fluorescent assays, colorimetric assays, enzymatic assays, or by any other means capable of detecting the presence or the absence of the complex. In another embodiment, the exposure and detection steps are iterated using different combinations of first and second probes derived from an array of a plurality of first probes and an array of a plurality of second probes.
In another embodiment, the invention is a method of priming an enzyme-catalyzed reaction such as polymerase chain reaction (PCR), primer extension, ligation, or other amplification methods. In this embodiment, the target nucleic acid is exposed to the first and second probes under conditions that permit the formation of a complex if the target sequence in the target nucleic acid is present. In this embodiment, an enzyme typically is provided to catalyze a reaction primed by the complex.
It should be understood that the order of the steps of the methods of the invention is immaterial so long as the invention remains operable, i.e. permits the formation of a multipartite binding moiety, its association with a target nucleic acid, and any subsequent operations or steps. Moreover, two or more steps may be conducted simultaneously.
The foregoing, and other features and advantages of the invention, as well as the invention itself, will be more fully understood from the following description, drawings, and claims.